During the past decades, enormous efforts have been dedicated to the establishment and culturing of plant-based systems for the accumulation and harvesting of native or heterologous proteins and secondary metabolites. The literature provides a vast quantity of evidential material that proves the utility of plant-based systems to produce a large variety of desired substances that are either secreted into the culture medium or isolated from the producing cells, tissues, organelles or even whole plants or parts thereof. Likewise, a broad range of transformation protocols exist that ensure the establishment of either stably or transiently transformed plant material. However, there is still a need for a reliable, relatively cost-efficient and rapid technology to obtain high yields of a desired product from plant cells.
It has been repeatedly reported that transformation of a population of plant cells such as a plant suspension culture frequently results in transgenic cultures that exhibit cells with highly heterogeneous (mixed) and inconsistent expression levels of the target protein related to the mixture of epigenetically different cells within the primary heterogeneous cell population. Within recombinant cell lines the heterogeneity in transgene expression demonstrates a serious problem in terms of production rates.
A main problem is that high-producing clones are often rare events within a transformation assay and it is very time consuming to establish a homogeneous high-producing cell line. A still ongoing technical challenge, therefore, is the elite transgenic event production and recovery from a freshly transformed or already transgenic plant culture.
For flow cytometric sorting such as e.g. FACS application, single spherical cells must be obtained from the usually aggregated plant cell population or culture by enzymatic digestion of the cells to liberate protoplasts. For most plant species, however, the regeneration of single protoplasts is hampered by the necessity to be maintained at certain population densities.
A reliable and reproducible procedure for the regeneration of a single transgenic cell/protoplast or for the regeneration of a whole fertile plant therefrom (especially after flow cytometric sorting) has hitherto not been described.